Expression systems for the production of recombinant polypeptides are well-known in the state of the art and are described by, e.g., Marino, M. H., Biopharm. 2 (1989) 18-33; Goeddel, D. V., et al., Methods Enzymol. 185 (1990) 3-7; Wurm, F., and Bernard, A., Curr. Opin. Biotechnol. 10 (1999) 156-159.
Polypeptides, such as antibodies and antibody fusions, for use in pharmaceutical applications are generally produced in mammalian cells such as CHO cells, NS0 cells, SP2/0 cells, COS cells, HEK cells, BHK cells, PER.C6™ cells, or the like. The elements of an eukaryotic expression plasmid are generally a prokaryotic plasmid propagation unit, for example for E. coli, comprising a prokaryotic origin of replication and a prokaryotic selection marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. For transient expression in mammalian cells a mammalian origin of replication, such as the SV40 Ori or OriP, can be included. As promoter a constitutive or inducible promoter can be selected. For optimized transcription a Kozak sequence may be included in the 5′ untranslated region. For mRNA processing, in particular mRNA splicing and transcription termination, mRNA splicing signals, depending on the organization of the structural gene (exon/intron organization), may be included as well as a polyadenylation signal.
Other polypeptides for use in pharmaceutical applications, e.g. insulin, interferon alpha-2, somatotropin, interleukin-2, GM-CSF and Reteplase, can be produced in prokaryotic cells, yeast, and mainly E. coli. The elements of an E. coli expression plasmid are generally an origin of replication, a selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest. An expression cassette generally comprises a promoter, a structural gene, and a transcription terminator. As promoter a constitutive or inducible promoter can be used. For optimized transcription a Shine-Dalgarno-Sequence or a variant thereof preceding the start codon of mRNA may be included in the 5′ untranslated region.